The goal of this work is to characterize membrane transporter genes in Cercospora fungi required for autoresistance to the photoactivated, active-oxygen-generating toxin cercosporin they produce for infection of host plants. Previous studies implicated a role for diverse membrane transporters in cercosporin resistance. In this study, transporters identified in a subtractive cDNA library between a Cercospora nicotianae wild type and a cercosporin-sensitive mutant were characterized, including two ABC transporters (CnATR2, CnATR3), an MFS transporter (CnMFS2), a uracil transporter, and a zinc transport protein. Phylogenetic analysis showed that only CnATR3 clustered with transporters previously characterized to be involved in cercosporin resistance. Quantitative RT-PCR analysis of gene expression under conditions of cercosporin toxicity, however, showed that only CnATR2 was upregulated, thus this gene was selected for further characterization. Transformation and expression of CnATR2 in the cercosporin-sensitive fungus Neurospora crassa significantly increased cercosporin resistance. Targeted gene disruption of CnATR2 in the wild type C. nicotianae, however, did not decrease resistance. Expression analysis of other transporters in the cnatr2 mutant under conditions of cercosporin toxicity showed significant upregulation of the cercosporin facilitator protein gene (CFP), encoding an MFS transporter previously characterized as playing an important role in cercosporin autoresistance in Cercospora species. We conclude that cercosporin autoresistance in Cercospora is mediated by multiple genes, and that the fungus compensates for mutations by up-regulation of other resistance genes. CnATR2 may be a useful gene, alone or in addition to other known resistance genes, for engineering Cercospora resistance in crop plants.

The CRG1 gene in Cercospora nicotianae encodes a transcription factor and is required for cercosporin toxin resistance and production. Cloning and sequencing of the downstream region of the CRG1 gene led to the discovery of an adjacent gene (PUT1) encoding a putative uracil transporter. Expression of CRG1 and PUT1 as assessed by Northern analysis indicated that, in addition to the expected monocistronic mRNAs (2.6 kb and 2.0 kb, respectively), a common 4.5-kb mRNA could be identified, using either a CRG1 or a PUT1 gene probe. The 2.6-kb transcript identified only by the CRG1 probe was expressed constitutively, whereas the 2.0-kb transcript identified only by the PUT1 probe was differentially expressed in various media. Four cDNA clones containing CRG1, PUT1, and the CRG1–PUT1 intergenic region were identified as part of the products from the 4.5-kb transcript. Both the 4.5-kb and 2.6-kb transcripts were not detectable in three crg1-disrupted mutants, using the CRG1 probe. The 2.0-kb transcript, but not the 4.5-kb one was detected using the PUT1 probe in the three crg1-disrupted mutants. Taken together, we conclude that the 4.5-kb transcript is a dicistronic mRNA of both CRG1 and PUT1 in the fungus C. nicotianae. This is the first example of a dicistronic mRNA identified in filamentous fungi.

Summary

A set of 53 cloned pea chloroplast DNA fragments representing approximately 90% of the chloroplast genome was used to probe Northern blots of total pea RNA, resulting in a nearly complete chloroplast transcription map. Similar analyses were performed for RNAs extracted from pea seedlings grown under several different light regimes. We have found that at least 85 kb of the 120 by pea chloroplast genome is represented as detectable transcripts. For many regions of the genome, we have detected multiple overlapping transcripts including both small, gene-sized RNAs and large transcripts covering entire gene clusters. All transcripts detected were more abundant (as a fraction of total cellular RNA) in light grown plants than in plants entirely in the dark. However, larger transcripts were generally more abundant in plants that had been exposed to only 24 h of white light (after germination in the dark) than in plants grown in continuous light. This study indicates that chloroplast genes are often grouped into multigene transcriptional units which can be cotranscribed, and that light-stimulated plastid development involves changes in the relative abundance of the overlapping RNAs of different length that result from transcription of these genes or gene clusters.

Summary

We have isolated and characterized the F0-ATPase subunit 6 gene (atp6) from tobacco mitochondria. The tobacco sequence exists as a single copy, is transcribed and contains an open reading frame (ORF) capable of encoding a peptide of 395 amino acids. The first 130 amino acids of the tobacco putative polypeptide show limited homology with the N terminus predicted for the maize ATPase subunit 6. Although poorly conserved at the sequence level, the tobacco and maize amino termini are hydrophilic and have a high percentage of charged amino acids. This portion of the predicted peptide may represent a presequence that is common to the ATPase subunit 6 of plants. Significant homology between tobacco and maize begins with amino acid 131, in a region that is highly conserved among fungal ATPase 6 subunits. In the remainder of the predicted protein, tobacco and maize share approximately 81% homology. A 41 by sequence and a 175 by conserved region found upstream from the tobacco atp6 coding region are homologous with sequence elements found in the 5′ flanking regions of other plant mitochondrial genes and may be important for regulation and expression of the atp6 gene.

Summary

Aberrant recombinations involving the mitochondrial atp9, atp6 and coxII genes have created unique chimeric sequences in the C male0sterile cytoplasm (cms-C) of maize. An apparent consequence of the rearrangements is the interchanging of transcriptional and/or translational regulatory signals for these genes, and alterations in the reading frames encoding the atp6 and coxII genes in the C cytoplasm. Particularly unusual is the organization of the atp6 gene in cms-C mitochondria, designated atp6-C. The atp6-C sequence is a triple gene fusion product comprised of DNAs derived from atp9, atp6 and an open reading frame of unknown origin. Although there is no direct evidence indicating that these chimeric genes are responsible for the cytoplasmic male sterility (cms) trait, their novel arrangements and the strong correlation between these genes and the C type of male sterility suggest such a role.

Summary

By direct RNA mapping, we have identified the precise 5′ end of the maize mitochondria) 26S rRNA. The 5′ termini of the 26S rRNA are 17 and 18 nt downstream from the 5′ end predicted by Dale et al. (1984) from homology between the maize rRNA and E. coli large subunit ribosomal RNA. In addition, we have discovered a larger 26S species, presumably a 26S precursor, and precisely mapped its 5′ end. The maize mitochondrial rRNA genes are probably regulated by control regions that differ from those already characterized in other genomes: the maize mitochondrial 26S, 18S and 5S rRNAs lack sequences that resemble the promoter regions of genes from other mitochondria and bacteria. However, the mature and precursor rRNAs all contain a tetranucleotide, AAUC, at their 5′ ends (AAAC in the 5S rRNA). The sequence is also seen in the same position in several other plant mitochondrial mRNAs. We propose therefore that AAUC is a transcription or processing signal which is possibly unique to plant mitochondria.

Summary

The plastid 16S–23S intergenic spacer region in Conopholis americana, a totally heterotrophic angiosperm in the family Orobanchaceae, has undergone large deletions, including the entire tRNAIle gene and all but small remnants of the tRNAAla gene. The length of the region is less than 20% of that of other land plants which have been investigated, making it the smallest 16S–23S intergenic spacer reported thus far for any land plant. The remaining sequences in the spacer are 90.1% identical to tobacco, indicating that, while the region is well conserved at the sequence level, it is evolving rapidly by deletion. Experiments using the polymerase chain reaction and hybridization to DNA gel blots have failed to revcal either of the two missing tRNA genes elsewhere in the Conopholis cell.

Classical genetic analyses are not possible with the phytopathogenic fungus Cercospora kikuchii since no sexual stage has been identified. To facilitate gene mapping and to develop an understanding of the genome organization of C. kikuchii, an electrophoretic karyotype has been obtained using contour-clamped homogeneous electric field gel electrophoresis (CHEF). Eight chromosomes, two of which migrate as a doublet, have been separated into seven bands ranging from 2.0 to 5.5 Mb. Using this determination of chromosome number and size, the total genome size of C. kikuchii is estimated to be 28.4 Mb. In addition, genes encoding tubulin, ribosomal DNA, and four previously isolated light-enhanced cDNAs from C. kikuchii were assigned to chromosomes by Southern-hybridization analysis of CHEF blots.

Summary

We have previously identified two large open reading frames, designated ORF13 and ORF25, in the Texas male-sterile cytoplasm (cms-T) of maize mitochondrial DNA (mtDNA). ORF13 is a single copy gene of chimeric origin that is uniquely transcribed and translated in the mitochondria of cms-T maize, where it produces a polypeptide of approximately 13,000 Mr. The ORF13 reading frame does not occur in the maize N, C or S cytoplasms or Nicotiana tabacum. ORF25 exists as a single copy and is transcribed in the four major maize cytoplasms (N, T, C and S) and N. tabacum. The predicted ORF25 polypeptide has a molecular weight of 24,374 in normal maize and 22,439 in tobacco. Several nucleotide and predicted amino acid changes have occurred in the ORF25 gene among the four maize cytoplasms and N. tabacum. Properties such as transcription and conservation of the sequence between two diverse species suggests that ORF25 encodes a functional plant mitochondrial gene. The ORF25 sequence of maize contains a chloroplast DNA insert homologous to a tRNA-Arg gene; this chloroplast DNA insert is absent in the tobacco ORF25 sequence. Comparison of the ORF25 and ORF13 sequences in restored and non-restored cms-T indicates no differences in their nucleotide sequences. Thus fertility restoration does not alter the primary sequences of ORF13 or ORF25.

RNA editing was analysed in the mitochondrial ATPase complex subunit 6 gene (atp6) transcripts of the C male-sterile cytoplasm (cms-C) of maize. The only copy of atp6 in cms-C, designated C-atp6, is a triple gene fusion product comprised of DNA sequences derived from atp9, atp6, and an unknown origin. Sequences of cDNAs revealed 19 C to U alterations resulting in 16 amino acid residue changes compared to the genomic sequence. The only C to U edit in the 39-nucleotide sequence similar to atp9 was comparable to a change in the complete atp9 mRNAs of Petunia, Oenothera, wheat, and sorghum. The 442 nucleotides of unknown origin were not edited. The 18 editing events within the atp6 homologous region were similar to those in the atp6 transcripts of sorghum. RNA editing in maize C-atp6 transcripts introduces a translational stop codon at the same position where it is created by editing in sorghum and Oenothera atp6 mRNAs and is already present in atp6 open reading frames of most other plant and non-plant organisms. Our results, along with other reports on editing in chimeric transcripts, indicate that RNA editing is not influenced by rearrangements but instead is sequence specific.